COVID-19 vaccination is the most effective strategy for preventing severe disease and death. Inactivated vaccines are the most accessible type of COVID-19 vaccines in developing countries. Several studies, including work from our group, have demonstrated that the third dose (booster vaccination) of inactivated COVID-19 vaccine induces robust humoral and cellular immune responses. The present study aimed to examine miRNA expression profile in participants who received a homologous third dose of the CoronaVac vaccine. Samples of peripheral blood mononuclear cells (PBMCs) were collected from healthcare volunteers both before and 1–2 weeks after the booster dose. miRNA microarray analysis in a discovery cohort of six volunteers identified 67 miRNAs with differential expression. Subsequently, the expression of six miRNAs related to immune responses was examined in a validation cohort of 31 participants via qRT-PCR. Our results validated the differential expression of miR-25-5p, miR-34c-3p, and miR-206 post-booster, with a significant correlation to the receptor binding domain (RBD)-specific antibody. Bioinformatic analysis suggested that miR-25-5p, miR-34c-3p, and miR-206 may target multiple pathways involved in immune regulation and inflammation. Therefore, our study highlights miR-25-5p, miR-34c-3p, and miR-206 in PBMCs as promising biomarkers for assessing the immune response induced by the booster dose of the CoronaVac vaccine.

Circulating extracellular vesicles (EVs) have been associated with the development of COVID-19 due to their roles in viral infection, inflammatory response, and thrombosis. However, the direct induction of lung inflammation by circulating EVs from severe COVID-19 patients remains unknown. EVs were extracted from the plasma of severe COVID-19 patients admitted to intensive care and healthy controls. To study the effect of COVID-19 EVs on lung inflammation, mice were intratracheally instilled with EVs. To examine the proinflammatory effects of EVs in vitro, bone marrow-derived macrophages were treated with EVs. COVID-19 but not control EVs triggered lung inflammation, as assessed by total protein level, total cell count, neutrophil count, and levels of proinflammatory cytokines in the bronchoalveolar lavage. COVID-19 EVs also promoted M1 polarization of alveolar macrophages in vivo. Treatment of bone marrow-derived macrophages with COVID-19 EVs enhanced the M1 phenotype and augmented the production of IL-1β, IL-6, and TNF-α. In summary, circulating EVs from severe COVID-19 patients induce lung inflammation in mice. EVs could become a potential therapeutic target for alleviating lung injury in COVID-19. IMPORTANCE:Extracellular vesicles (EVs) have been reported to facilitate cytokine storm, coagulation, vascular dysfunction, and the spread of the virus in COVID-19. The direct role of circulating EVs from severe COVID-19 patients in lung injury remains unrecognized. Our study demonstrated that plasma EVs obtained from severe COVID-19 patients induced lung inflammation and polarization of alveolar macrophages in vivo. In vitro experiments also revealed the proinflammatory effects of COVID-19 EVs. The present study sheds fresh insight into the mechanisms of COVID-19-induced lung injury, highlighting EVs as a potential therapeutic target in combating the disease.

BACKGROUND:Macrophages are involved in various immune inflammatory disease conditions. This study aimed to investigate the role and mechanism of macrophages in regulating acute intestinal injury in neonatal necrotizing enterocolitis (NEC).METHODS:CD68, nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3), cysteine aspartate-specific protease-1 (caspase-1), and interleukin-1β (IL-1β) in paraffin sections of intestinal tissues from NEC and control patients were detected with immunohistochemistry, immunofluorescence, and western blot. Hypertonic pet milk, hypoxia and cold stimulation were used to establish a mouse (wild type and Nlrp3-/-) model of NEC. The mouse macrophage (RAW 264.7) and rat intestinal epithelial cell-6 lines were also cultured followed by various treatments. Macrophages, intestinal epithelial cell injuries, and IL-1β release were determined.RESULTS:Compared to the gut "healthy" patients, the intestinal lamina propria of NEC patients had high macrophage infiltration and high NLRP3, caspase-1, and IL-1β levels. Furthermore, in vivo, the survival rate of Nlrp3-/- NEC mice was dramatically improved, the proportion of intestinal macrophages was reduced, and intestinal injury was decreased compared to those of wild-type NEC mice. NLRP3, caspase-1, and IL-1β derived from macrophages or supernatant from cocultures of macrophages and intestinal epithelial cells also caused intestinal epithelial cell injuries.CONCLUSIONS:Macrophage activation may be essential for NEC development. NLRP3/caspase-1/IL-1β cellular signals derived from macrophages may be the underlying mechanism of NEC development, and all these may be therapeutic targets for developing treatments for NEC.

Septic shock as a subset of sepsis, has a much higher mortality, while the mechanism is still elusive. This study was aimed at identifying core mechanisms associated with septic shock and its high mortality by investigating transcriptome data. We screened 72 septic-shock-associated genes (SSAGs) with differential expression between septic shock and sepsis in the discovery dataset. Further gene set enrichment analysis identified upregulated neutrophil activation and impaired T-cell activation in septic shock. Co-expression analysis revealed nine co-expressed gene modules. In addition, we determined twenty-one prognostic SSAGs using cox regression analysis in an independent dataset. Moreover, protein-protein interaction (PPI) network revealed two clusters. Among these neutrophil activation was enriched in the most positively-related modules and the cluster2 PPI network, while T-cell activation was enriched in both the most negatively-related module and one of the most positively-related modules as well as the cluster1 PPI network. ELANE, LCN2 and IFI44 were identified as hub genes with CytoHubba methods and semantic similarity analysis. Notably, ELANE was the only prognostic gene and was further validated in an external dataset. Blood neutrophil count was demonstrated to increase in septic shock and be a risky factor of prognosis based on clinical data. In conclusions, septic shock is associated with upregulated neutrophil activation and dysregulated T-cell activation. Three hub genes might have potentials as sensitive markers for the further translational research and ELANE could be a robust prognostic biomarker and effective therapeutic target.

ObjectiveThe incidence of intraoperative hypothermia remains high in pediatric patients during anesthesia and surgery even though core body temperature monitoring and warming systems have been greatly improved in recent years. We analyzed the risk factors and outcomes of intraoperative hypothermia in neonates and infants undergoing general anesthesia and surgery.MethodsThe data on the incidence of intraoperative hypothermia, other clinical characteristics, and outcomes from electronic records of 1,091 patients (501 neonates and 590 infants between 28 days and 1 year old), who received general anesthesia and surgery, were harvested and analyzed. Intraoperative hypothermia was defined as a core temperature below 36°C during surgery.ResultsThe incidence of intraoperative hypothermia in neonates was 82.83%, which was extremely higher than in infants (38.31%, p < 0.001)—the same as the lowest body temperature (35.05 ± 0.69°C vs. 35.40 ± 0.68°C, p < 0.001) and the hypothermia duration (86.6 ± 44.5 min vs. 75.0 ± 52.4 min, p < 0.001). Intraoperative hypothermia was associated with prolonged PACU, ICU, hospital stay, postoperative bleeding, and transfusion in either age group. Intraoperative hypothermia in infants was also related to prolonged postoperative extubation time and surgical site infection. After univariate and multivariate analyses, the age (OR = 0.902, p < 0.001), weight (OR = 0.480, p = 0.013), prematurity (OR = 2.793, p = 0.036), surgery time of more than 60 min (OR = 3.743, p < 0.001), prewarming (OR = 0.081, p < 0.001), received >20 mL/kg fluid (OR = 2.938, p = 0.004), and emergency surgery (OR = 2.142, p = 0.019) were associated with hypothermia in neonates. Similar to neonates, age (OR = 0.991, p < 0.001), weight (OR = 0.783, p = 0.019), surgery time >60 min (OR = 2.140, p = 0.017), pre-warming (OR = 0.017, p < 0.001), and receive >20 mL/kg fluid (OR = 3.074, p = 0.001) were relevant factors to intraoperative hypothermia in infants along with the ASA grade (OR = 4.135, p < 0.001).ConclusionThe incidence of intraoperative hypothermia was still high, especially in neonates, with a few detrimental complications. Neonates and infants each have their different risk factors associated with intraoperative hypothermia, but younger age, lower weight, longer surgery time, received more fluid, and no prewarming management were the common risk factors.

BackgroundDuring the COVID-19 epidemic, vaccination has become the most safe and effective way to prevent severe illness and death. Inactivated vaccines are the most widely used type of COVID-19 vaccines in the world. In contrast to spike-based mRNA/protein COVID-19 vaccines, inactivated vaccines generate antibodies and T cell responses against both spike and non-spike antigens. However, the knowledge of inactivated vaccines in inducing non-spike-specific T cell response is very limited.MethodsIn this study, eighteen healthcare volunteers received a homogenous booster (third) dose of the CoronaVac vaccine at least 6 months after the second dose. CD4+ and CD8+ T cell responses against a peptide pool from wild-type (WT) non-spike proteins and spike peptide pools from WT, Delta, and Omicron SARS-CoV-2 were examined before and 1-2 weeks after the booster dose. ResultsThe booster dose elevated cytokine response in CD4+ and CD8+ T cells as well as expression of cytotoxic marker CD107a in CD8+ T cells in response to non-spike and spike antigens. The frequencies of cytokine-secreting non-spike-specific CD4+ and CD8+ T cells correlated well with those of spike-specific from WT, Delta, and Omicron. Activation-induced markers (AIM) assay also revealed that booster vaccination elicited non-spike-specific CD4+ and CD8+ T cell responses. In addition, booster vaccination produced similar spike-specific AIM+CD4+ and AIM+CD8+ T cell responses to WT, Delta, and Omicron, indicting strong cross-reactivity of functional cellular response between WT and variants. Furthermore, booster vaccination induced effector memory phenotypes of spike-specific and non-spike-specific CD4+ and CD8+ T cells. ConclusionsThese data suggest that the booster dose of inactive vaccines broadens both non-spike-specific and spike-specific T cell responses against SARS-CoV-2.

Objective:To explore the regulation of group 2 innate lymphoid cells (ILC2) recruitment by interleukin-33 (IL-33) , to examine the effect of ILC2 on macrophages during peritoneal infection in sepsis and to further elucidate the immune regulatory mechanism of peritoneal infection.Methods:Forty healthy 6 to 8 week-old male C57BL/6 wild-type mice, twenty Il-33 -/- mice on a C57BL/6 background, and ten IL-33 receptor ST2 knockout (Il1rl1 -/-) mice were bred and housed in the SPF Medical Animal Center of Zhejiang University and the University of Pittsburgh, USA. Receipts of sham surgery were designated as controls and underwent identical surgical procedures were septic mice. IL-33 protein was administered to rmIL-33 group via an exogenous intraperitoneal injection and the changes of ILC2 and its intracellular cytokine levels in peritoneal lavage fluid were detected by flow cytometry. The effects of IL-9 and IL-13 secreted by ILC2 on phagocytic ability of macrophages were confirmed by in vitro experiments. Statistical analysis was performed with t-test or one-way ANOVA test. Results:Compared with ILC2 before/after IL-33 stimulation, the proportion of ILC2 in Lin-CD45 + cells of abdominal cavity increased [(9.90±0.80) vs. (18.43±0.46) ]and the proportion of ILC2 in abdominal cavity jumped after IL-33 stimulation and the difference was statistically significant ( P<0.001). The number of ILC2 in abdominal cavity was [(5 786.00±289.50) vs. (17 820.00±324.30) ]and the number of ILC2 spiked in abdominal cavity after IL-33 stimulation and the difference was statistically significant ( P<0.001). C57BL/6 wild-type mice had a marked increase of peritoneal ILC2 recruitment as compared with control group at 24 h postoperatively [(20.50±0.59) vs. (10.83±1.01) ]and the difference was statistically significant ( P=0.001). However, ILC2 in peritoneal cavity of Il-33 -/- and Il1rl1 -/- mice showed no change at 24 h postoperatively. Moreover, the proportion of ILC2 in Il-33 -/- mice were significantly increased after IL-33 stimulation compared to non-stimulation group (10.53±0.47 vs. 19.43±0.87, P<0.001) , so as in the CLP induced septic mice (10.13±0.57 vs. 21.37±0.86, P<0.001). The study of cytokine levels secreted by ILC2 recruited from abdominal cavity showed that the levels of IL-9 and IL-13 secreted by ILC2 in abdominal cavity increased markedly at 12 h postoperatively and the contents of IL-9 before/after operation were [(16.53±0.74) vs. (24.67±1.45) ]and the differences were statistically significant ( P= 0.007). The proportions of IL-13 at pre/post-operation were [(15.40±0.87) vs. (29.40±0.95) ]and the differences were statistically significant ( P<0.001) while the contents of IL-4 were [(6.14±0.59) vs. (5.89±1.05) ]and the differences were not statistically significant ( P=0.836). An intraperitoneal injection of recombinant IL-33 protein promoted the recruitment of CD45 +F4/80 + macrophages in peritoneal cavity and the proportions before/after injection were [(52.40±1.70) vs. (72.40±2.02) ]and the difference was statistically significant ( P=0.002). Under the action of IL-9/13 secreted by ILC2, phagocytic ability of macrophages was markedly elevated as compared with control group. Phagocytic ability of macrophages under the action of IL-9 was [(61.27±1.46) vs. (48.30±1.65) ]and phagocytic ability of macrophages under the action of IL-13 was [(61.63±2.03) vs. (48.30±1.65) ]and the difference was statistically significant ( P=0.007). Conclusions:IL-33 promotes ILC2 and macrophage recruitment during peritoneal sepsis. And the recruitment of peritoneal ILC2 depends on IL-33/ST2 signaling pathway. Peritoneal ILC2 derived cytokines IL-9 and IL-13 enhance the phagocytosis of macrophages.